Pyridyl-triazine derivatives as microbiocidal active substances

ABSTRACT

Triazine derivatives of formula (1) wherein R 1  is C 1 –C 20 alkyl; C 3 –C 7 cycloalkyl; or C 1 –C 20 perfluoroalkyl; R 2  is hydrogen; C 1 –C 20 alkyl; or C 3 –C 7 cycloalkyl; and R 3  is hydrogen; C 1 –C 20 alkyl; C 3 –C 7 cycloalky; C 1 –C 20 perfluoroalky; C 1 C 20 alkyl-carbonyl; C 3 –C 7 cycloalkyl-carbonyl; C 1 –C 20 perfluoroalkyl-carbonyl; or phenylcarbonyl; are described. The compounds exhibit pronounced action against gram-positive and gram-negative bacteria and also against yeasts and moulds. They are also suitable for treating or preventing biofilms on human tooth surfaces and oral mucosa

The present invention relates to selected2-alkyl-4-amino-6-pyridyl-1,3,5-triazine derivatives, to the preparationof such compounds, and to the use of such compounds in the antimicrobialtreatment of surfaces, as antimicrobial active substances againstgram-positive and gram-negative bacteria, yeasts and fungi and also inthe preservation of cosmetics, household products, textiles and plasticsand for use in disinfectants.

The compounds according to the invention correspond to formula

-   R₁ is C₁–C₂₀alkyl; C₃–C₇cycloalkyl; or C₁–C₂₀perfluoroalkyl;-   R₂ is hydrogen; C₁–C₂₀alkyl; or C₃–C₇cycloalkyl; and-   R₃ is hydrogen; C₁–C₂₀alkyl; C₃–C₇cycloalkyl; C₁–C₂₀perfluoroalkyl;    C₁–C₂₀alkyl-carbonyl; C₃–C₇cycloalkyl-carbonyl;    C₁–C₂₀perfluoroalkyl-carbonyl; or phenylcarbonyl.

C₁–C₂₀Alkyl radicals are straight-chain or branched alkyl radicals suchas, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,tert-butyl, amyl, isoamyl or tert-amyl, hexyl, 2-ethylhexyl, heptyl,octyl, isooctyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, pentadecyl,hexadecyl, heptadecyl, octadecyl or eicosyl.

C₃–C₇Cycloalkyl is, for example, cyclopropyl, cyclobutyl, cyclopentyl,cycloheptyl and, especially, cyclohexyl. Those radicals may besubstituted, for example by one or more identical or differentC₁–C₄alkyl radicals, especially by methyl, and/or by hydroxy. Whencycloalkyl radicals are substituted by one or more substituents, theyare preferably substituted by one, two or four, especially one or two,identical or different substituents.

The invention relates preferably to compounds of formula (1) wherein

-   R₁ is C₁–C₄alkyl;-   R₂ is hydrogen; and-   R₃ is C₆–C₂₀alkyl; C₃–C₇cycloalkyl; C₁–C₂₀perfluoroalkyl;    C₁–C₂₀alkyl-carbonyl; C₃–C₇cyclo-alkyl-carbonyl; or    C₁–C₂₀perfluoroalkyl-carbonyl.

Very special preference is given to compounds of formula (1) wherein

-   R₁ is C₁–C₄alkyl;-   R₂ is hydrogen; and-   R₃ is C₂–C₆alkyl; C₁–C₁₂perfluoroalkyl; C₁–C₁₂alkyl-carbonyl; or    C₁–C₁₂perfluoroalkyl-carbonyl.

Very special preference is given to compounds according to the inventionthat correspond to formula

wherein

-   R₁ is C₁–C₄alkyl; especially methyl; or tert-butyl; and-   R₃ is C₆–C₂₀alkyl; C₂–C₆alkyl; C₃–C₇cycloalkyl;    C₁–C₂₀perfluoroalkyl; C₁–C₂₀alkyl-carbonyl;    C₃–C₇cycloalkyl-carbonyl; or C₁–C₂₀perfluoroalkyl-carbonyl;    or to formula

wherein

-   R₁ is C₁–C₄alkyl; especially methyl; and-   R₃ is C₆–C₂₀alkyl; C₃–C₇cycloalkyl; C₁–C₂₀perfluoroalkyl;    C₁–C₂₀alkyl-carbonyl; or C₁–C₂₀perfluoroalkyl-carbonyl;    or to formula

wherein

-   R₁ is C₁–C₄alkyl; especially tert-butyl; and-   R₃ is C₆–C₂₀alkyl; C₃–C₇cycloalkyl; C₁–C₂₀perfluoroalkyl;    C₁–C₂₀alkyl-carbonyl;C₃–C₇cyclo-alkyl-carbonyl; or    C₁–C₂₀perfluoroalkyl-carbonyl.

Very special preference is given to compounds of formula (2) wherein

-   R₁ is tert-butyl; and-   R₃ is C₆–C₂₀alkyl; especially octyl.

Preparation of the triazine derivatives according to the invention iscarried out using processes known per se and, depending on the basicstructure, may be carried out in accordance with one or more of themethods described hereinbelow.

Synthesis of the triazines of the general formula (1) wherein R₁=methylis preferably carried out as follows: first, pyridylamidines known fromthe literature (for example, from Medwid, J. Med. Chem., 1990 (33).1230), where appropriate in the form of their salts, are reacted withcyano-imidates, which have been prepared in accordance with data in theliterature, for example as described in WO/0014056, or which arecommercially available, preferably cyano-methyl-imidates of formula(1c), in an inert solvent, preferably methanol, to form theamino-methyl-triazines of formula (1d). The latter compounds are then,in accordance with processes known from the literature (for example,Beckwith, The Chemistry of Amides, Wiley, 1970 (86)), acylated withcarboxylic acids or derivatives thereof, preferably with carboxylicanhydrides, to form the N-acyl-amino-triazines of formula (1e), whichare subsequently reduced using reducing agents such as, for example,hydrogen, simple or complex metal hydrides, transition metals or saltsthereof, but preferably using borane, to form theN-alkyl-amino-triazines of formula (1a).

The reaction sequence can be illustrated by the following Scheme:

In another process, 2-alkyl-triazines can be prepared by condensation ofN-acylated imino esters with substituted guanidines. For that purpose,pyridylimino esters of formula (1f), which have been prepared inaccordance with data In the literature or which are commerciallyavailable, are acylated in accordance with customary methods, and theresulting N-acyl-imino esters of formula (1g) are reacted with mono- ordi-substituted guanidines or salts thereof in an inert solvent,preferably isopropanol or tert-butanol, to form the pyridyl-triazines offormula (1a):

The invention relates also to the processes for the preparation of the2-alkyl-4-amino-6-pyridyl-1,3,5-triazine derivatives according to theinvention.

The triazine derivatives of formula (1) according to the inventionexhibit pronounced antimicrobial action, especially againstgram-positive and gram-negative bacteria and against bacteria of theskin flora, and also against yeasts and moulds. They are accordinglysuitable especially for disinfection, deodorisation, and for general andantimicrobial treatment of the skin and mucosa and of integumentaryappendages (hair), more especially for the disinfection of hands andwounds.

They are accordingly suitable as antimicrobial active substances andpreservatives in personal care preparations such as, for example,shampoos, bath additives, haircare preparations, liquid and solid soaps(based on synthetic surfactants and salts of saturated and/orunsaturated fatty acids), lotions and creams, deodorants, other aqueousor alcoholic solutions, e.g. cleansing solutions for the skin, moistcleansing cloths, oils or powders.

The invention accordingly relates also to a personal care preparationcomprising at least one compound of formula (1) and cosmeticallytolerable carriers or adjuvants.

The personal care preparation according to the invention contains from0.01 to 15% by weight, preferably from 0.1 to 10% by weight, based onthe total weight of the composition, of a compound of formula (1), andcosmetically tolerable adjuvants.

Depending upon the form of the personal care preparation, it comprises,in addition to the triazine derivative of formula (1), furtherconstituents such as, for example, sequestering agents, colorants,perfume oils, thickeners or solidifiers (consistency regulators),emollients, UV-absorbers, skin protective agents, antioxidants,additives that improve the mechanical properties, such as dicarboxylicacids and/or aluminium, zinc, calcium or magnesium salts of C₁₄–C₂₂fattyacids, and, optionally, preservatives.

The personal care preparation according to the invention may beformulated as a water-in-oil or oil-in-water emulsion, an alcoholic oralcohol-containing formulation, a vesicular dispersion of an ionic ornon-ionic amphiphilic lipid, a gel, a solid stick or an aerosolformulation.

As a water-in-oil or oil-in-water emulsion, the cosmetically tolerableadjuvant contains preferably from 5 to 50% of an oil phase, from 5 to20% of an emulsifier and from 30 to 90% water. The oil phase maycomprise any oil suitable for cosmetic formulations such as, forexample, one or more hydrocarbon oils, a wax, a natural oil, a siliconeoil, a fatty acid ester or a fatty alcohol. Preferred mono- or poly-olsare ethanol, isopropanol, propylene glycol, hexylene glycol, glyceroland sorbitol.

Cosmetic formulations according to the invention are used in variousfields. There come into consideration, for example, especially thefollowing preparations:

-   -   skin-care preparations, e.g. skin-washing and cleansing        preparations in the form of tablet-form or liquid soaps,        synthetic detergents or washing pastes,    -   bath preparations, e.g. liquid (foam baths, milks, shower        preparations) or solid bath preparations, e.g. bath cubes and        bath salts;    -   skin-care preparations, e.g. skin emulsions, multi-emulsions or        skin oils;    -   cosmetic personal care preparations, e.g. facial make-up in the        form of day creams or powder creams, face powder (loose or        pressed), rouge or cream make-up, eye-care preparations, e.g.        eyeshadow preparations, mascaras, eyeliners, eye creams or        eye-fix creams; lip-care preparations, e.g. lipsticks, lip        gloss, lip contour pencils, nail-care preparations, such as nail        varnish, nail varnish removers, nail hardeners or cuticle        removers;    -   intimate hygiene preparations, e.g. intimate washing lotions or        intimate sprays;    -   foot-care preparations, e.g. foot baths, foot powders, foot        creams or foot balsams, special deodorants and antiperspirants        or callus-removing preparations;    -   light-protective preparations, such as sun milks, lotions,        creams or oils, sun-blocks or tropicals, pre-tanning        preparations or after-sun preparations;    -   skin-tanning preparations, e.g. self-tanning creams;    -   depigmenting preparations, e.g. preparations for bleaching the        skin or skin-lightening preparations;    -   insect-repellents, e.g. insect-repellent oils, lotions, sprays        or sticks;    -   deodorants, such as deodorant sprays, pump-action sprays,        deodorant gels, sticks or roll-ons;    -   antiperspirants, e.g. antiperspirant sticks, creams or roll-ons;    -   preparations for cleansing and caring for blemished skin, e.g.        synthetic detergents (solid or liquid), peeling or scrub        preparations or peeling masks;    -   hair-removal preparations in chemical form (depilation), e.g.        hair-removing powders, liquid hair-removing preparations, cream-        or paste-form hair-removing preparations, hair-removing        preparations in gel form or aerosol foams;    -   shaving preparations, e.g. shaving soap, foaming shaving creams,        non-foaming shaving creams, foams and gels, preshave        preparations for dry shaving, aftershaves or aftershave lotions;    -   fragrance preparations, e.g. fragrances (eau de Cologne, eau de        toilette, eau de parfum, parfum de toilette, perfume), perfume        oils or perfume creams;    -   dental care, denture-care and mouth-care preparations, e.g.        toothpastes, gel toothpastes, tooth powders, mouthwash        concentrates, anti-plaque mouthwashes, denture cleaners or        denture fixatives;    -   cosmetic hair-treatment preparations, e.g. hair-washing        preparations in the form of shampoos and conditioners, hair-care        preparations, e.g. pretreatment preparations, hair tonics,        styling creams, styling gels, pomades, hair rinses, treatment        packs, intensive hair treatments, hair-structuring preparations,        e.g. hair-waving preparations for permanent waves (hot wave,        mild wave, cold wave), hair-straightening preparations, liquid        hair-setting preparations, hair foams, hairsprays, bleaching        preparations, e.g. hydrogen peroxide solutions, lightening        shampoos, bleaching creams, bleaching powders, bleaching pastes        or oils, temporary, semi-permanent or permanent hair colorants,        preparations containing self-oxidising dyes, or natural hair        colorants, such as henna or camomile.

An antimicrobial soap has, for example, the following composition:

-   0.01 to 5% by weight of a compound of formula (1)-   0.3 to 1% by weight titanium dioxide,-   1 to 10% by weight stearic acid,-   soap base ad 100%, e.g. a sodium salt of tallow fatty acid or    coconut fatty acid, or glycerol.

A shampoo has, for example, the following composition:

-   0.01 to 5% by weight of a compound of formula (1),-   12.0% by weight sodium laureth-2-sulfate,-   4.0% by weight cocamidopropyl betaine,-   3.0% by weight NaCl and-   water ad 100%.

A deodorant has, for example, the following composition:

-   0.01 to 5% by weight of a compound of formula (1),-   60% by weight ethanol,-   0.3% by weight perfume oil, and-   water ad 100%.

The invention relates also to an oral composition containing from 0.01to 15% by weight, based on the total weight of the composition, of acompound of formula (1), and orally tolerable adjuvants.

Example of an Oral Composition:

-   10% by weight sorbitol,-   10% by weight glycerol,-   15% by weight ethanol,-   15% by weight propylene glycol,-   0.5% by weight sodium lauryl sulfate,-   0.25% by weight sodium methylcocyl taurate,-   0.25% by weight polyoxypropylene/polyoxyethylene block copolymer,-   0.10% by weight peppermint flavouring,-   0.1 to 0.5% by weight of a compound of formula (1), and-   48.6% by weight water.

The oral composition according to the invention may be, for example, inthe form of a gel, a paste, a cream or an aqueous preparation(mouthwash).

The oral composition according to the invention may also comprisecompounds that release fluoride ions which are effective against theformation of caries, for example inorganic fluoride salts, e.g. sodium,potassium, ammonium or calcium fluoride, or organic fluoride salts, e.g.amine fluorides, which are known under the trade name Olafluor.

The triazine derivatives of formula (1) according to the Invention arealso suitable for treating, especially preserving, textile fibrematerials. Such materials are undyed and dyed or printed fibrematerials, for example of silk, wool, polyamide or polyurethanes, andespecially cellulosic fibre materials of all kinds. Such fibre materialsare, for example, natural cellulose fibres, such as cotton, linen, juteand hemp, as well as cellulose and regenerated cellulose. Preferredsuitable textile fibre materials are made of cotton.

The triazine derivatives of formula (1) according to the invention aresuitable also for treating plastics, especially imparting antimicrobialproperties to or preserving plastics, such as, for example,polyethylene, polypropylene, polyurethane, polyester, polyamide,polycarbonate, latex etc. Fields of use therefor are, for example, floorcoverings, plastics coatings, plastics containers and packagingmaterials; kitchen and bathroom utensils (e.g. brushes, shower curtains,sponges, bathmats), latex, filter materials (air and water filters),plastics articles used in the field of medicine such as, for example,dressing materials, syringes, catheters etc., so-called “medicaldevices”, gloves and mattresses.

Paper, for example papers used for hygiene purposes, may also beprovided with antimicrobial properties using the triazine derivatives offormula (1) according to the invention.

It is also possible for nonwovens such as, for example, nappies/diapers,sanitary towels, panty liners, and cloths for hygiene and householduses, to be provided with antimicrobial properties in accordance withthe invention.

The triazine derivatives of formula (1) are also used in washing andcleaning formulations such as, for example, liquid or powder washingagents or softeners.

The triazine derivatives of formula (1) can also be used especially inhousehold and general-purpose cleaners for cleaning and disinfectinghard surfaces.

A cleaning preparation has, for example, the following composition:

0.01 to 5% of a compound of formula (1) 3.0% octyl alcohol 4EO 1.3%fatty alcohol C₈–C₁₀ polyglucoside 3.0% isopropanol water ad 100%.

In addition to preserving cosmetic and household products, thepreservation of technical products, the provision of technical productswith antimicrobial properties and use as a biocide in technicalprocesses are also possible, for example in paper treatment, especiallyin paper treatment liquors, printing thickeners of starch or cellulosederivatives, surface-coatings and paints.

The triazine derivatives of formula (1) according to the invention arealso suitable for the antimicrobial treatment of wood and for theantimicrobial treatment of leather, the preserving of leather and theprovision of leather with antimicrobial properties.

The triazine derivatives of formula (1) according to the invention arealso suitable for the protection of cosmetic products and householdproducts from microbial damage.

In addition to their generally antimicrobial action, the triazinederivatives of formula (1) according to the invention are moreovercapable of penetrating biofilms on living and non-living surfaces, ofpreventing the adhesion of bacteria to surfaces and any further build-upof the biofilm, of detaching such build-up, and/or of inhibiting thefurther growth of the biofilm-forming micro-organisms in the biologicalmatrix or of killing such micro-organisms.

Biofilms are understood, very generally, to be aggregations of livingand dead micro-organisms, especially bacteria, that adhere to living andnon-living surfaces, In conjunction with their metabolites in the formof extracellular polymeric substances (EPS matrix), e.g.polysaccharides. The activity of antimicrobial substances that normallyexhibit a pronounced growth-inhibiting or lethal action with respect toplanktonic cells may be greatly reduced with respect to micro-organismsthat are organised in biofilms, for example because of inadequate activesubstance penetration into the biological matrix.

In the present invention, this relates, very especially, to biofilms onhuman tooth surfaces and oral mucosa, which play a crucial role in theonset of degenerative diseases in the oral cavity, e.g. caries orperiodontitis, as a result of the micro-organisms that are formed ortheir metabolites.

The following Examples illustrate, but do not limit, the presentinvention.

EXAMPLE 1 Synthesis of 2-amino-4-methyl-6-pyrid-2-yl-1,3,5-triazine

Reaction Scheme:

A solution of 17.0 g (0.32 mol) of sodium methoxide in 60 ml of methanolis added dropwise, at room temperature, to a solution of 50.0 g (0.32mol) of pyrid-2-ylamidine hydrochloride in 240 ml of methanol. Afterstirring for 20 minutes, filtration is carried out and the solution isthen added dropwise to 31.4 g (0.32 mol) of methyl-N-cyanoethanimidate,with stirring. After stirring for 2 hours at room temperature, coolingto 0° C. is carried out and the precipitate is subsequently filteredoff.

The compound of formula (101) is purified by re-crystallisation fromethanol.

Yield: 30.0 g (50% of theory)

¹H NMR (CDCl₃): 7.35–8.75 (m,4H,arom. H), 6.00 (s,2H,NH₂), 2.50(s,3H,CH₃)

¹³C NMR (CDCl₃): 177.8/171.0/167.7 (triazine C's),153.9/150.4/137.7/126.4/124.6 (pyridine C's), 26.0 (CH₃)

m/z=187 (GC/MS)

Synthesis of the homologous 3-pyridyl derivative of formula

is carried out analogously.

¹H NMR (DMSO-d₆): 8.20–9.50 (m,4H,arom. H), 8.15 (s,2H,NH₂), 2.45(s,3H,CH₃)

¹³C NMR (DMSO-d₆): 174.8/166.0/165.2 (triazine C's),145.3/144.1/142.3/133.8/127.6 (pyridine C's), 24.5 (CH₃)

m/z=187 (GC/MS)

EXAMPLE 2 Preparation of2-acetylamino-4-methyl-6-pyrid-2-yl-1,3,5-triazine

Reaction Scheme

1.9 g (10 mmol) of formula (102) from Example 1 are stirred in 5 ml ofacetanhydride for 12 hours at 100° C. After concentration, the residueis taken up in methylene chloride and washed with NaHCO₃ solution. Thecompound of formula (103) is obtained quantitatively and in a puritythat is sufficient for the subsequent reaction (>95% GC).

¹H NMR (CDCl₃): 7.41–8.81 (m,4H, arom. H), 8.42 (s,1H,NH), 2.68(s,3H,CH₃), 2.58 (s,3H,CH₃)

¹³C NMR (CDCl₃): 179.4/171.61171.4/164.1 (triazine C's/CO),153.0/150.7/137.7/127.0/125.0 (pyridine C's), 26.5 (CH₃), 26.2 (CH₃)

The following N-acylamino-triazines are obtained analogously (Table 1):

TABLE 1 Compound Method of Purity of formula Structure analysis [%(FID)] (104)

GC-MS 47 (105)

GC-MS 73 (106)

GC-MS 70 (107)

GC-MS 82 (108)

GC-MS 58 (109)

GC-MS 81 (110)

GC-MS 70 (111)

GC-MS 80 (112)

GC-MS/LC-MS 30 (113)

GC-MS 89 (114)

GC-MS 92 (115)

GC-MS 20 (116)

GC-MS 59 (117)

GC-MS 70 (118)

GC-MS 90 (119)

GC-MS 72 (120)

GC-MS 87 (121)

GC-MS 97 (122)

GC-MS 79 (123)

GC-MS 42 (124)

GC-MS 90 (125)

GC-MS 86 (126)

GC-MS 35

EXAMPLE 3 General Synthesis of2-alkylamino-4-methyl-6-pyridyl-1,3,5-triazines

To 1 mmol of the acylamino-triazine in 1 ml of toluene there is added,at 0° C., the stoichiometric amount of a 2M solution of borane-dimethylsulfide complex in toluene, and heating at reflux is then carried outfor 4 hours. After cooling to room temperature, the reaction solution istreated with 10% Na₂CO₃ solution, dried and concentrated by evaporation.The identity of the reaction products is confirmed by GC/MS or LC/MS andthe compounds are used for screening against micro-organisms withoutfurther purification. The following compounds are obtained in thatmanner:

TABLE 2 Compound Method of Purity of formula Structure analysis [%(FID)] (127)

GC-MS 69 (128)

GC-MS 30 (129)

GC-MS 66 (130)

GC-MS 90 (131)

GC-MS 60 (132)

GC-MS 100 (133)

GC-MS 100 (134)

GC-MS 100 (135)

GC-MS 40 (136)

GC-MS 30 (137)

GC-MS 92 (138)

GC-MS 35 (139)

GC-MS 57

EXAMPLE 4 Synthesis of2-tert-butyl-4-n-octylamino-6-pyrid-2-yl-1,3,5-triazine (Compound ofFormula (140))

2.60 g (21.5 mmol) of pivaloyl chloride in 10 ml of toluene are slowlyadded to 2.66 g (19.5 mmol) of 2-pyridylimidomethyl ester and 2.17 g(21.5 mmol) of NEt₃ in 30 ml of toluene at 0° C. After stirring for 24hours at room temperature, filtration is carried out and the solvent isdrawn off. The residue is taken up in 10 ml of tert-butanol, and asolution of 6.61 g (30 mmol) of n-octylguanidine in 20 ml tert-butanolis then added.

After 24 hours at room temperature, the solvent is drawn off and thecompound of formula (140) is purified by chromatography on silica gel(ethyl acetate/hexane 9/1).

Yield: 5.1 g (77% of theory)

¹H NMR (CDCl₃): 7.33–8.73 (m,4H,arom. H), 5.79 (s,1H,NH), 1.54(m,2H,CH₂), 1.20–1.33 (m,21H, CH₂/C(CH₃)₃), 0.81 (t,3H,CH₃)

¹³C NMR (CDCl₃): 186.3/169.7/166.8 (triazine C's),154.7/150.2/137.3/125.9/124.7 (pyridyl C's), 39.7/29.2 (C(CH₃)₃), 14.5(CH₃), 23.0–41.3 (CH₂)

m/z=341

EXAMPLE 5 Determination of the Minimum Inhibitory Concentration (MIC) inthe Agar Incorporation Test (MIC Test)

Medium: Casein/soymeal peptone agar (Merck) *Sabouraud 4% glucose agar(Merck) Diluent: Sterile 0.85% NaCl solution Test organisms:Staphylococcus aureus ATCC 6538 Staphylococcus epidermidis ATCC 12228Corynebacterium xerosis ATCC 373 C. minutissimum ATCC 23348Propionibacterium acnes ATCC 6919 Escherichia coli ATCC 10536Escherichia coli NCTC 8196 Proteus vulgaris ATCC 6896 Klebsiellapneumoniae ATCC 4352 Salmonella choleraesuis ATCC 9184 Pseudomonasaeruginosa ATCC 15442 Candida albicans ATCC 10231 Aspergillus niger ATCC6275 Incubation: 24 hours at 37° C. *3 days at 28° C. Test solution: 1%stock solutions of all the test substances are prepared in a suitablesolvent and diluted in serial dilutions to end concentrations of from1000 ppm to 10 ppm. Test principle: 0.3 ml of each dilution step ismixed with 15 ml of nutrient medium while the latter is still liquid.After the nutrient medium has solidified, 10 μl of a suitable organismdilution of each of the test strains in 0.85% NaCl solution are spottedonto the agar medium: Staphylococcus aureus 1:100 ATCC 6538Staphylococcus epidermidis 1:1000 ATCC 12228 Corynebacterium xerosis1:1000 ATCC 373 C. minutissimum ATCC 1:10 dilution 23348Propionibacterium acnes 1:10 dilution ATCC 6919

The plates are incubated at 37° C. for 24 hours (A. niger at 28° C. for3 days) and then the highest dilution (lowest concentration) of the testsubstance at which growth is just no longer discernible (corresponds tothe MIC) is determined.

The results are shown in Tables 3 and 4.

TABLE 3 MIC in [ppm] Compound of formula (140) Staphylococcus aureusATCC 6538 0.94 Staphylococcus epidermidis ATCC 12228 120 Corynebacteriumxerosis ATCC 373 0.94 C. minutissimum ATCC 23348 0.94 Propionibacteriumacnes ATCC 6919 0.94 Escherichia coli ATCC 10536 60 Escherichia coliNCTC 8196 60 Proteus vulgaris ATCC 6896 >120 Klebsiella pneumoniae ATCC4352 120 Salmonella choleraesuis ATCC 9184 120 Pseudomonas aeruginosaATCC 15442 >120 Candida albicans ATCC 10231 >120 Aspergillus niger ATCC6275 >120

TABLE 4 MIC in [ppm] Compound of formula Staphylococcus aureus ATCC 6538(123) 8 (131) 30 (135) 60

EXAMPLE 6 Testing the Substantivity of2-tert-butyl-4-n-octylamino-6-pyrid-2-yl-1,3,5-triazine (140) onHydroxyapatite and Determination of the Growth-Inhibiting Action

Principle:

Hydroxyapatite discs (diameter 10 mm) are placed in artificial saliva(Deutsche Zahnärztliche Zeitschrift DZZ 5/2002) for 4 hours, rinsed 4times in NaCl solution and then incubated for 30 minutes in an ethanolicsolution of the test substance (140) at 5 ppm. The discs are thentransferred to a Nunclon-surface titre plate (12 wells) and incubated at37° C. for 24 hours in CASO nutrient medium inoculated with Actinomycesviscosus ATCC 43146 (about 10⁵/ml).

Result:

FIG. 1 shows the inhibition of the growth of Actinomyces viscosus by thetest substance (140) following adsorption of the substance ontohydroxyapatite discs pre-treated with artificial saliva, compared to anuntreated control. The substantivity is dependent upon presence of thesaliva.

EXAMPLE 7 Testing the Activity of2-tert-butyl-4-n-octylamino-6-pyrid-2-yl-1,3,5-triazine (140) in theBiofilm Model

Principle: See Guggenheim et al. (2001), “Validation of an in vitrobiofilm model of supragingival plaque”, J. Dent Res. 80 (1).

Hydroxyapatite discs are pre-treated with human saliva and a biofilm ofa defined mixed culture of various gram+ and gram− oral micro-organisms(Streptococci, Actinomyces sp., Veillonella & Fusobacterium sp. andothers) and a yeast (Candida albicans) is allowed to grow on thehydroxyapatite. Over the course of the test, which lasts a total of 4days, the discs are immersed several times in an application formulationof the substance (0.5% (140)). Finally, the biofilm is harvested and theliving cell count is determined.

Result:

The results are shown in Table 3. The triazine compound causes a clearreduction in the microbial count of the biofilm on the hydroxyapatitediscs, compared to the placebo formulation without active substance anda control containing 10% ethanol. A comparison substance, which exhibitsno corresponding action, was also tested.

TABLE 3 Control Comparison Compound of Placebo Sample (10% ethanol)substance formula (140) formulation (Mean) 4.9 × 10⁸ 1.6 × 10⁷ 1.1 × 10⁵1.4 × 10⁷ [CPU/ disc]

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the inhibition of the growth of Actinomyces viscosus by thetest substance (140) following adsorption of the substance ontohydroxyapatite discs pre-treated with artificial saliva, compared to anuntreated control.

1. A compound of formula

wherein R₁ is C₁–C₄alkyl; and R₃ is C₆–C₂₀alkyl; C₃–C₇cycloalkyl;C₁–C₂₀perfluoroalkyl; C₁–C₂₀alkyl-carbonyl; C₃–C₇cycloalkyl-carbonyl; orC₁–C₂₀perfluoroalkyl-carbonyl.
 2. A compound according to claim 1,wherein R₁ is tert-butyl; and R₃ is C₆–C₂₀alkyl.
 3. A compound offormula

wherein R₁ is C₁–C₄alkyl; and R₃ is C₆–C₂₀alkyl; C₃–C₇cycloalkyl;C₁–C₂₀perfluoroalkyl; C₁–C₂₀alkyl-carbonyl; C₃–C₇cycloalkyl-carbonyl; orC₁–C₂₀perfluoroalkyl-carbonyl.
 4. A compound of formula

R₁ is C₁–C₄alkyl; and R₃ is C₆–C₂₀alkyl; C₃–C₇cycloalkyl;C₁–C₂₀perfluoroalkyl; C₁-C₂₀alkyl-carbonyl; C₃–C₇cycloalkyl-carbonyl; orC₁–C₂₀perfluoroalkyl-carbonyl.